Safety and immunogenicity of booster vaccination and fractional dosing with Ad26.COV2.S or BNT162b2 in Ad26.COV2.S-vaccinated participants

We report the safety and immunogenicity of fractional and full dose Ad26.COV2.S and BNT162b2 in an open label phase 2 trial of participants previously vaccinated with a single dose of Ad26.COV2.S, with 91.4% showing evidence of previous SARS-CoV-2 infection. A total of 286 adults (with or without HIV) were enrolled >4 months after an Ad26.COV2.S prime and randomized 1:1:1:1 to receive either a full or half-dose booster of Ad26.COV2.S or BNT162b2 vaccine. B cell responses (binding, neutralization and antibody dependent cellular cytotoxicity-ADCC), and spike-specific T-cell responses were evaluated at baseline, 2, 12 and 24 weeks post-boost. Antibody and T-cell immunity targeting the Ad26 vector was also evaluated. No vaccine-associated serious adverse events were recorded. The full- and half-dose BNT162b2 boosted anti-SARS-CoV-2 binding antibody levels (3.9- and 4.5-fold, respectively) and neutralizing antibody levels (4.4- and 10-fold). Binding and neutralizing antibodies following half-dose Ad26.COV2.S were not significantly boosted. Full-dose Ad26.COV2.S did not boost binding antibodies but slightly enhanced neutralizing antibodies (2.1-fold). ADCC was marginally increased only after a full-dose BNT162b2. T-cell responses followed a similar pattern to neutralizing antibodies. Six months post-boost, antibody and T-cell responses had waned to baseline levels. While we detected strong anti-vector immunity, there was no correlation between anti-vector immunity in Ad26.COV2.S recipients and spike-specific neutralizing antibody or T-cell responses post-Ad26.COV2.S boosting. Overall, in the context of hybrid immunity, boosting with heterologous full- or half-dose BNT162b2 mRNA vaccine demonstrated superior immunogenicity 2 weeks post-vaccination compared to homologous Ad26.COV2.S, though rapid waning occurred by 12 weeks post-boost. Trial Registration: The study has been registered to the South African National Clinical Trial Registry (SANCTR): DOH-27-012022-7841. The approval letter from SANCTR has been provided in the up-loaded documents.


Introduction
The development of vaccines against SARS-CoV-2 was unparalleled, with numerous platforms suited to rapid production dominating use in initial vaccination programs globally.This included adenovirus-vectored vaccines, such as the Janssen Ad26.COV2.S, replicationincompetent adenovirus 26 vectored SARS-CoV-2 spike protein vaccine.This vaccine is registered as a single dose, with subsequent boosters recommended.This was the first vaccine available nationally in South Africa, and health care workers, and later other essential workers, were offered this vaccine as part of the Sisonke trial [1].The mRNA-based Pfizer BNT162b2 Comirnaty vaccine became available in South Africa subsequently as part of the national vaccine rollout.
However, given the waning of humoral and cellular immunity over time, COVID-19 vaccine boosting may be beneficial, especially in individuals at high risk for severe disease.The choice of booster, timing and dose remain largely dependent on regulatory and national considerations, including fiscal constraints and capacity of the health care system.Several studies have demonstrated a more robust humoral and cellular immune response with a heterologous boost compared to a homologous boost, in particular when boosting is with an mRNA vaccine [12,15,23].A further consideration is that due to pressure on vaccine development, cost and equitable access, which are significantly impacted if multiple boosters are needed, strategies such as fractional dosing should be considered.Fractional dosing has previously been used with other vaccines such as yellow fever [24].
Vaccines which use a viral vector to deliver the immunogen may be inhibited by pre-existing immunity to the virus on which the vector is based [42,43].In addition to eliciting immunity to the vaccine target, vaccination with an adenovirus vectored vaccine has been shown to elicit neutralizing antibody and T-cell immunity to the vector itself [44][45][46].This may reduce the ability of repeated doses of the vectored vaccine to infect cells or reduce vaccine vectorinfected cell survival.The degree of elicited anti-vector immunity could therefore potentially determine the effectiveness of vectored immunization in a population with previous immunity to the virus on which the vector is based, or effectiveness if the vaccinees have been previously immunized with the same vector.However additional factors, including transgene persistence as well as local or systemic persistence may also impact the effectiveness of viral vectored vaccines.
In this study, we evaluated the immunogenicity and safety of diverse boost strategies after primary Ad26.COV2.S vaccination.We tested fractional and full dose heterologous and homologous booster Ad26.COV2.S and BNT162b2 vaccinations.We also determined the effects of HIV status, and degree of anti-adenovirus 26 vector immunity before and after boosting.We observed that heterologous boosting of Ad26.COV2.S with the BNT162b2 mRNA vaccine was the most effective regimen to transiently enhance humoral and T-cell immunity and HIV status did not have a substantial effect on the outcome.We show that .homologous boosting with Ad26.COV2.S occurred in the context of high anti-Ad26 vector immunity.Although, Ad26.COV2.S gave a weak increase in antibody and cellular responses against SARS-CoV-2 spike, we observed no correlation between vaccine response and anti-Ad26 neutralizing antibody levels, as previously reported [47,48].
. Procedures.Study visits took place at baseline (randomization), 2 weeks, 12 weeks and 24 weeks.At each visit, medical history, COVID-19 infection history (symptoms or positive test) and vaccination history (COVID-19 vaccine or other vaccination) was taken, followed by a targeted clinical examination when necessary.At the baseline visit, participants received a single booster vaccination, as per the randomization arm.Diary cards were issued and participants were trained to collect data up to 7 days post booster.Telephonic contact at day 7 was conducted to enquire about severity or ongoing nature of reactogenicity.Blood samples were drawn at each visit and included HIV testing at baseline (in all except those known to be PLWH), CD4 count and HIV viral load (in PLWH), full blood count (BL, W2 and additional visits in PLWH), D-dimers (BL and W2).A nasopharyngeal swab for SARS-CoV-2 PCR was conducted at baseline.Interim visits were held for any safety concerns or if a participant had COVID-19 symptoms or a positive SARS-CoV-2 PCR or antigen test outside of the study.

Safety. Reactogenicity was collected and graded according to criteria included in Food and
Drug Administration (FDA) "The Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials" [49].All grade 3 reactogenicity events, SAEs, SUSARs and adverse drug reactions were reported.A Data Safety and Monitoring Committee was established to evaluate protocol-defined safety events and immunogenicity data.
Cells were incubated for 1 h.Wells were then filled with 3 mL complete growth medium.After 4 days of infection (completion of passage 1), cells were trypsinized, centrifuged and resuspended in 4 mL growth medium.Then, all infected cells were added to Vero E6 cells (CRL-1586, ATCC).The coculture of ACE2-expressing H1299-E3 and Vero E6 cells was incubated for 4 days.The viral supernatant from this culture (passage 2 stock) was used for experiments.
H1299-E3 cells were plated at 30,000 cells/well 1 day pre-infection.Aliquots of cryo-preserved plasma samples were heat-inactivated and clarified by centrifugation.Virus stocks were used at approximately 50-100 focus-forming units per microwell and added to diluted plasma.
Antibody-virus mixtures were incubated for 1 h at 37°C.Cells were infected with 100 μL of the virus-antibody mixtures for 1 h, then 100 μL of a RPMI 1640 (Sigma-Aldrich), 1.5% carboxymethylcellulose (Sigma-Aldrich) overlay was added without removing the inoculum.
Cells were fixed 18 h post-infection.Foci were stained with a rabbit anti-spike monoclonal .antibody (0.5 μg/mL, BS-R2B12, GenScript) overnight at 4°C, washed and then incubated with a horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody (1 μg/mL, Abcam ab205718) for 2 h.TrueBlue peroxidase substrate (SeraCare) was then added and incubated for 20 min.Plates were imaged in an ImmunoSpot Ultra-V S6-02-6140 Analyzer ELISPOT instrument with BioSpot Professional built-in image analysis (C.T.L).All statistics and fitting were performed using custom code in MATLAB v.2019b.Neutralization data were fit to: Tx=1/1+(D/ID50).Tx is the number of foci normalized to the number of foci in the absence of plasma on the same plate at dilution D and ID50 is the plasma dilution giving 50% neutralization.FRNT50 = 1/ID50.Values of FRNT50 <1 are set to 1 (undiluted), the lowest measurable value.As, the most concentrated plasma dilution was 1:25, FRNT50 <25 were extrapolated.To calculate confidence intervals, FRNT50 or fold-change in FRNT50 per participant was log-transformed and arithmetic mean plus and minus two standard deviations were calculated for the log transformed values.These were exponentiated to obtain the upper and lower 95% confidence intervals on the geometric mean FRNT50 or the fold-change in FRNT50 geometric means.

SARS-CoV-2 spike and nucleocapsid enzyme-linked immunosorbent assay (ELISA)
For ELISA, Hexapro SARS-CoV-2 full spike protein with the D614G substitution were expressed in Human Embryonic Kidney (HEK) 293F suspension cells by transfecting the cells with the respective expression plasmid.After incubating for 6 days at 37°C, proteins were first purified using a nickel resin followed by size exclusion chromatography.Relevant fractions were collected and frozen at -80°C until use.Two µg/mL of D614G spike or nucleocapsid protein was used to coat 96-well, high-binding plates (Corning) and incubated overnight at 4°C.The plates were incubated in a blocking buffer consisting of 1x PBS, 5% skimmed milk powder, 0.05% Tween 20.Plasma samples were diluted to 1:100 starting dilution in a blocking buffer and added to the plates.IgG secondary antibody (Merck) was diluted to 1:3000 in blocking buffer and added to the plates followed by TMB substrate (Thermofisher Scientific).
Upon stopping the reaction with 1 M H2SO4, absorbance was measured at 450 nm.For spike ELISA, mAbs CR3022 was used as a positive control and Palivizumab was used as a negative control.

Lentiviral pseudovirus production and neutralization assay
Virus production and pseudovirus neutralization assays were done as previously described.
. Luminescence was measured using PerkinElmer Life Sciences Model Victor X luminometer.Neutralization was measured as described by a reduction in luciferase gene expression after single-round infection of 293T/ACE2.MF cells with spike-pseudotyped viruses.Titers were calculated as the reciprocal plasma dilution (ID50) causing 50% reduction of relative light units.

Antibody-dependent cellular cytotoxicity (ADCC) assay
The ability of plasma antibodies to cross-link FcγRIIIa (CD16) and spike expressing cells was measured as a proxy for ADCC as previously described.HEK293T cells were transfected with 5 μg of SARS-CoV-2 wild-type variant spike (D614G), Beta, Delta and Omicron BA.1 spike plasmids using PEI-MAX 40,000 (Polysciences) and incubated for 2 days at 37°C.Expression of spike was confirmed by binding of CR3022 and P2B-2F6 and their detection by anti-IgG APC (Biolegend) measured by flow cytometry.Subsequently, 1x10 5 spike-transfected cells per well were incubated with heat inactivated plasma (1:100 final dilution) or control mAbs (final concentration of 100 μg/mL) in RPMI 1640 media supplemented with 10% FBS and 1% Pen/Strep (R10; Gibco) for 1 h at 37°C.Jurkat-Lucia™ NFAT-CD16 cells (Invivogen) (2x10 5 cells/well) were added and incubated for 24 h at 37°C.Twenty μl of supernatant was then transferred to a white 96-well plate with 50 μl of reconstituted QUANTI-Luc secreted luciferase and read immediately on a Victor 3 luminometer with 1s integration time.Cells were gated on singlets, live cells (determined by Live/dead™ Viability dye; Thermofisher Scientific), and those cells that were positive for IgG and spike specific monoclonal antibodies binding to their surface.Relative light units (RLU) of a no antibody control were subtracted as background.
Palivizumab was used as a negative control, while CR3022 was used as a positive control, and P2B-2F6 to differentiate the Beta from the D614G variant.To induce the transgene, 1x cell stimulation cocktail (Thermofisher Scientific) and 2 μg/ml ionomycin in R10 was added as a positive control.

Outcomes
The primary objectives of the study were to evaluate safety and reactogenicity and humoral and cellular immunogenicity to full and half dose homologous and heterologous booster vaccinations, at each study visit.Primary endpoint measures included measuring nucleocapsid binding antibody titers, neutralization titers and T-cell response magnitudes.Safety was measured by participant self-report using diary cards and graded according to FDA standards [49].The primary immunogenicity endpoint was defined as any study arm eliciting <75% of the highest geometric mean titre (GMT) response in the study.Together with the DSMB, the study team used this criteria to determine whether a subsequent booster should be offered to participants, and which booster if so.In this study, based on results, all participants except those who received the full dose BNT162b2 vaccination, were offered a full dose BNT162b2 booster in addition to that received according to their original randomisation.

Statistical Analysis
A two-tailed Wilcoxon signed-rank test or a Friedman test with Dunn's correction was used to assess statistical differences between paired samples.A Mann-Whitney test or a Kruskal-Wallis test with Dunn's corrections was used to compare multiple groups.Correlations were tested by a two-tailed non-parametric Spearman's rank test.In all cases, P values of less than or equal to 0.05 were considered significant.

Data Sharing
Protocol and data underlying the findings described in this manuscript may be obtained from the lead authors upon request.
Participants received the booster vaccine at a median of 271 days (IQR: 255-296) after the Ad26.COV2.S prime.Nine breakthrough infections (BTI) were confirmed by SARS-CoV-2 PCR during the trial and all were mild infections that resolved within 4-12 days, 7/9 in the Ad26.COV2.S arms.Three BTI occurred between W2 and W12, and the remainder between W12 and W24 (median: 17.1 weeks post boost).
No events of thrombosis with thrombocytopenia syndrome (TTS) were reported; thrombocytopenia was reported in three participants, two had thrombocytopaenia at baseline.There were 14 serious AEs (SAEs) on study, non-related to study product, and no AEs of special interest (AESIs).

Antibody responses after booster vaccination
We evaluated spike binding antibody titers at baseline (BL) and week 2 (W2), week 12 (W12) and week 24 (W24) post-boost in the four vaccination arms using a trimeric spike ELISA for ancestral D614G virus.Relatively high spike binding antibody titers were detected in all groups at BL (geometric mean titer (GMT), EC 50: >1000; Figure 3A).This is consistent with our observation that the majority of participants showed evidence of prior infection (Table 1).
Spike antibody titers in the half-and full-dose Ad26.COV2.S arms were not significantly boosted by W2 (1.15 and 1.29 fold change in GMT, respectively), and did not change significantly up to W24.In contrast, half-and full-dose BNT162b2 arms demonstrated a significant increase in binding antibody titers at W2 (3.9 and 4.54 fold change, respectively), to .a GMT >4500 (Figure 3A).Spike binding antibodies in the BNT162b2 arms reduced to half that of W2 titers by W12, and had returned to BL levels by W24.These kinetics were reflected in a significantly higher fold-change between W2 and BL in the BNT162b2 arms compared to the Ad26.COV2.S arms (Figure 3B).Although waning had occurred by W12, EC50 titers in the BNT162b2 arms were still significantly higher than the Ad26.COV2.S arms (Figure 3C).While some differences between the arms persisted at W24, relatively high antibody titers, similar to those observed at BL and prior to a boost, were noted in all four arms, with GMT of 1016, 1140, 1563 and 1686 for half-dose Ad26.COV2.S, full-dose Ad26.COV2.S, half-dose BNT162b2 and full-dose BNT162b2, respectively.
Using a live virus neutralization assay, we evaluated neutralizing antibody titers to the ancestral D614G virus and Omicron BA.5, which was the most recent dominant sub-lineage at the time of W24 collection.At BL, neutralization activity against the D614G virus was similar between the arms (GMT FRNT50 values of 546, 345, 393 and 334 for half-dose Ad26.COV2.S, full-dose Ad26.COV2.S, half-dose BNT162b2 and full-dose BNT162b2, respectively; Figure 4A).When measured at W2, half-dose Ad26.COV2.S did not boost BL neutralizing titers, while full-dose Ad26.COV2.S led to a 2.14-fold increase (Figure 4B).Both doses of BNT162b2 demonstrated a superior ability to boost neutralizing antibodies at W2 (8.5 to 10.2fold compared to BL), with all but one participant increasing neutralizing titer (Figure 4B).As expected from its known immune evasion properties, FRNT50 values for BA.5 neutralization were considerably lower than for ancestral virus (GMT of 84, 75, 84 and 63 for the four arms, respectively; Figure 4C).The kinetics mirrored those for D614G neutralization, with both doses of BNT162b2 demonstrating enhanced boosting of neutralizing antibodies compared to Ad26.COV2.S (Figure 4D).By week 24, neutralizing titers had waned significantly for the D614G virus, to a GMT of 732 and 840 for the BNT162b2 half-and full-doses, which was significantly higher than full-dose Ad26.COV2.S, with a GMT of 292 (Figure 4E).Neutralization of BA.5 was poor at W24 (GMT of , with no differences between the .groups.Five participants with documented BTI between W2 and W24 (Figure 4A,C, red lines) showed boosted neutralization activity, as expected.
In parallel, we assessed neutralizing antibody titers against an expanded panel of virus variants, namely D614G, Beta, Delta, Omicron BA.1 and BA.4/5 (which share identical spikes) using a lentiviral pseudovirus assay, limited to HIV-negative participants in the cohort.The data were consistent with our observations from the live virus assay, where significant boosting of neutralizing antibodies occurred at W2 against all variants for BNT162b2, but not for either the half-or full-dose Ad26.COV2.S (Figure 5).Titers against all variants declined from the W2 peak by W12, with a 2.4 to 5.7-fold drop, and levels remained constant to W24, with BNT162b2 arms trending to higher titers, regardless of the variant at W24.Finally, we investigated antibody-dependent cellular cytotoxicity (ADCC) responses against the D614G, Beta, Delta and BA.1 spikes (Figure 6A and 6B) at BL and 2 weeks post-boost.
As observed for the binding and neutralizing antibody responses, there was no increase in titers at W2 for either the half-or full-dose Ad26.COV2.S arms (median fold change 0.97 and 0.91, respectively), consistent with the fact that binding antibodies and ADCC potential are generally correlated.However, the half-dose BNT162b2 failed to trigger increased ADCC (median change 1.01), in contrast to the binding and neutralization results.For the full-dose BNT162b2 we observed marginally but nevertheless significantly higher ADCC titers against D614G, Beta and BA.1 spike at W2 compared to BL, but not to the same extent as binding or neutralizing responses.

T-cell responses before and after booster vaccination
We also measured SARS-CoV-2 spike-specific T-cell responses before and after homologous and heterologous vaccination with full or half-dose vaccines (n=214).Prior to boosting, spikespecific CD4+ responses were detected in most participants (>94.4%), with a frequency comparable between the four arms (Figure 7A).Two weeks after booster immunization, the .frequency of spike-specific CD4+ T cells was significantly increased in participants who received BNT162b2 compared to BL (median: 0.23% vs 0.12% for half dose BNT162b2 and 0.25% vs 0.11% for full dose BNT162b2, respectively), while in participants boosted with Ad26.COV2.S, the median frequency of SARS-CoV-2 spike-specific CD4+ T cells, while statistically higher than BL, demonstrated only a marginal increase (0.12% vs 0.10% for half dose and 0.15% vs 0.11% for full dose) (Figure 7B).The median fold change in CD4+ response between BL and W2 was significantly greater after a BNT162b2 booster (1.8 for half dose and 1.98 for full dose) compared to an Ad26.COV2.S booster (1.1 for both half and full dose; Figure 7C).Overall, ~80% of participants boosted with BNT162b2 (regardless of the dose) had an increased spike-specific CD4+ T-cell response, while only 35.3% of participants who received half dose Ad26.COV2.S and 43.4% in those receiving a full dose Ad26.COV2.S booster expanded their CD4+ T-cell responses (Figure 7D).Spike-specific CD8+ T-cell responses were less frequent than CD4+ responses, detected in only ~55% of the participants at BL, and for those with a CD8+ response, the frequency of spike-specific CD8+ T cells was comparable between the four groups at BL (Figure 7E).Two weeks after boosting, a significant increase in the median CD8+ T-cell response was observed after a full-dose of Ad26.COV2.S (p = 0.002) and both half-or full-dose of BNT162b2 (p = 0.003 and p <0.001, respectively; Figure 7F).Assessing only participants with a spike CD8+ response at both time points, the median fold change at W2 post-boost was significantly higher after a BNT162b2 booster (1.91 for half-dose and 2.24 for full-dose) compared to an Ad26.COV2.S booster (1.22 for half-dose and 1.24 for full-dose; Figure 7G).However, it is important to note that individual responses were highly variable within all groups, with spike-specific CD8+ responses contracting, remaining negative or expanding.Overall, approximately half of participants boosted with BNT162b2 displayed an increase in spike-specific CD8+ T-cell responses, whereas an expansion of the CD8+ response was observed in only 1/3 of participants who received a half-dose Ad26.COV2.S booster and ~40% in those receiving a full-dose Ad26.COV2.S booster (Figure 7H).

The durability of T-cell responses was then assessed by measuring spike-specific T cells 24
weeks after the booster vaccination in a subset of participants (n=190).Pairwise comparison of spike-specific CD4+ T-cell frequencies at W24 relative to W2 demonstrated a significant contraction in the CD4+ response for all groups (p <0.001 for both BNT162b2 boosters and half-dose Ad26.COV2.S and p = 0.0047 for full-dose Ad26.COV2.S), subsiding to BL levels (Figure 8A).Consequently, 24 weeks post-boosting, the magnitude of spike-specific CD4+ T cells was comparable in all trial arms (Figure 8B).In contrast, the median frequency of spikespecific CD8+ T cells appeared to increase between W2 and W24 in the Ad26.COV2.S group, although individual responses were highly variable, with some participants showing substantial waning or stable levels while others demonstrated gain of a CD8+ T-cell response that could be related to an undocumented BTI (Figure 8C).Overall, as for the CD4+ T-cell response, the frequency of spike-specific CD8+ T cells was similar across the four vaccine regimens when examined 24 weeks after booster vaccination (Figure 8D).

PLWH develop comparable antibody and T-cell responses after boosting
To determine whether HIV infection impacts vaccine booster responsiveness, participants were stratified by their HIV status.Prior to boosting, significantly lower titers of both binding and live virus neutralizing antibodies were found in PLWH who were viremic (viral load >200 HIV-1 mRNA copies/mL, n = 10) compared to virally suppressed individuals, who had similar antibody profiles to HIV-negative individuals (Figure 9A-B).Examination of baseline T-cell responses also revealed deficiencies, where significantly fewer viremic PLWH displayed spike-specific T-cell responses compared to aviremic and HIV-negative participants at BL (50% vs 100% and 97.6% for CD4+ and 10% vs 58.2% and 57.6% for CD8+; Figure 9C-D).
Two weeks after booster vaccination, there was no difference in the degree of boosting (as measured by median fold-change between W2 and BL) for binding or neutralizing antibody titers between PLWH and HIV-negative participants for all vaccination groups (Figure 9E-F).
For T-cell responses, however, the median fold change in CD4+ response in the full dose BNT162b2 group was significantly lower in PLWH compared to the HIV-negative participants  At W24, comparable titers of binding and neutralizing antibodies were detected between PLWH and HIV-negative participants in all vaccination groups, and T-cell frequencies did not show any deficiencies in PLWH (Figure 9I-L).In fact, significantly higher frequencies of spikespecific CD4+ T cells were observed in PLWH in some vaccination groups (Figure 9K), possibly reflecting persistence of the higher baseline CD4+ T-cell responses observed in PLWH.Overall, these data suggest that some differences in the degree of boosting in PLWH, but by 24 weeks after revaccination, the frequencies of binding and neutralizing antibodies as well as spike-specific CD4+ and CD8+ T cells were comparable between HIV-negative participants and PLWH.This suggests that SARS-CoV-2 specific responses persist to a similar extent in those with well-controlled HIV infection compared to HIV-negative individuals.
A proportion of viremic individuals, however, may be at risk of impaired T-cell responses and less durable antibody responses over the long term.

Ad26.COV2.S to boost spike-specific responses
We hypothesized that the muted capacity of Ad26.COV2.S to boost spike responses, compared to BNT162b2, may be due to anti-vector immunity, given that prior studies demonstrated that pre-existing Ad5 vector-specific nAbs and T cells have the capacity to limit responses to the transgene immunogen [44][45][46].To measure anti-Ad26 neutralizing antibody activity, we established an Ad26 neutralization assay in which dilutions of plasma from vaccinees were mixed with the Ad26.COV2.S vaccine, and vector infection was measured by spike expression in the Ad26 vector infected cells (Figure 10A-B).To quantify Ad26-specific neutralizing activity before and after boosting, we titrated participant plasma to obtain the plasma concentration needed to inhibit 50% of cellular infections with the Ad26 vector represented as ID50, the reciprocal of this plasma dilution (Figure 10C).We tested 24 participants who had received full dose Ad26.COV2.S, and compared them to 24 participants who were boosted with BNT162b2, where no expansion of the vector-specific response would be expected.At BL, the majority of participants in both groups had Ad26-specific neutralizing antibody responses above the level of quantification (Figure 10D).Two weeks later, there was a significant increase in vector-specific ID50 in the Ad26.COV2.S boosted group (p = 0.0006), but no boosting in the group receiving BNT162b2 (median fold-change in nAb ID50 between W2 and BL: 1.83 for Ad26.COV2.S and 0.87 for BNT162b2; Figure 10E).To determine the level of neutralizing immunity against Ad26.COV2.S before primary Ad26.COV2.S vaccination, we investigated an independent cohort where participants were vaccinated with Ad26.COV2.S for the first time, with a subset accessing a second Ad26.COV2.S vaccine (Supplementary Table 1).The levels of anti-Ad26 neutralizing immunity prior to Ad26.COV2.S prime vaccination were below the level of quantification in all but one participant.Titers were significantly higher after an Ad26.COV2.S prime (p <0.0001; Figure 10F) but did not further increase after a boost, and were of similar magnitude to those in the BaSiS trial (Figure 10F).
Finally, we investigated the association between Ad26-specific nAb titer at BL and the foldchange in spike neutralizing titer from W2 to BL, but found no correlation (Figure 10G).
To measure Ad26-specific T-cell responses, we stimulated PBMC with a pool of peptides spanning the viral hexon and penton proteins of Ad26 and characterized T-cell cytokine responses by flow cytometry.Ad26-specific T cells were readily detectable at BL and 2 weeks post-boosting (Figure 11A).We tested 44 participants who had received full-dose Ad26.COV2.S, and compared them to 20 participants who were boosted with BNT162b2, as a control group.At baseline, all participants in both groups had a CD4+ T-cell response to Ad26 (Figure 11B).Two weeks after Ad26.COV2.S booster vaccination, there was no overall increase in the Ad26-specific CD4+ response, which resembled the group who received the BNT162b2 booster.When comparing the fold-difference in the response from BL to W2, there .was a minor increase in the Ad26.COV2.S-boosted group (1.08 for Ad26.COV2.S and 0.91 for BNT162b2), reflecting a proportion of participants who had a marginal increase in the frequency of Ad26-specific CD4+ T cells (Figure 11C).We also investigated an independent unvaccinated healthcare worker cohort prior to Ad26.COV2.S vaccination (Supplementary Table 2); cross-sectional analysis indicated a significant, 3-fold higher magnitude of Ad26specific CD4+ T-cell responses 4 weeks after vaccination (p = 0.017; Figure 11D), similar to what we observed for Ad26 nAb responses.Interestingly, 85% of participants had an existing adenovirus CD4+ response even before the priming dose, which is the likely result of conserved T-cell cross-reactivity due to infection with other adenoviral types.
Similarly, we characterized CD8+ T-cell responses specific for Ad26.These were detectable in 75% of participants at baseline, and unlike CD4+ responses, were boosted significantly (p = 0.002) by a second dose of Ad26.COV2.S, demonstrating an increase in response of 1.2 compared to 0.95 for the BNT162b2-boosted group (Figure 11E and F).CD8+ responses were more rare in the cohort not previously vaccinated with Ad26.COV2.S, detectable in only 35% of unvaccinated participants.These were significantly higher (p = 0.005) and detectable in 81.8% of individuals 4 weeks after initial Ad26.COV2.S vaccination, at a magnitude and range similar to the baseline responses in our trial (Figure 11G).Importantly, and consistent with Ad26-specific nAb responses, there was no association between the magnitude of Ad26specific CD4+ or CD8+ T cells and spike-specific T-cell responses, either at BL or W2 (Figure 11H).Overall, anti-vector humoral and cellular immunity was abundantly detectable in our trial participants, but neither appeared to have a clear effect on dampening spike-specific responses. .

Discussion
In this trial we evaluated the safety and immunogenicity of fractional and full doses of the Janssen adeno-vectored Ad26.COV2.S vaccine and the Pfizer BNT162b2 mRNA vaccine in an open label phase 2 trial of adults who had previously received a single dose of Ad26.COV2.S.The overwhelming majority of participants had detectable anti-nucleocapsid responses prior to receiving their boosts, indicating prior SARS-CoV-2 infection and boosting on the background of extensive hybrid immunity.We investigated safety and immunogenicity in both HIV-negative participants and PLWH, with the latter group generally well-controlled with VL suppression, and only 13.8% of PLWH being viremic (VL >200 cps/ml).Safety profiles in all four booster regimens were similar, with no regimen resulting in appreciable increased reactogenicity beyond localized responses at the injection site and mild, transient symptoms such as headache and weakness.Documented BTI, while few, mostly occurred in the Ad26.COV2.S boosted participants.
Heterologous boosting with the BNT162b2 mRNA vaccine was superior to homologous vaccination at early time points (2-12 weeks) by multiple humoral and cellular immune measures, including binding and neutralizing antibodies, and CD4+ T-cell responses to SARS-CoV-2 spike.This result is consistent with other studies showing better neutralizing antibody and T-cell responses after heterologous relative to homologous boosting of Ad26.COV2.S with an mRNA vaccine [23,50].Similar results were observed with mRNA vaccine heterologous boosting of the ChAdOx1 chimpanzee adeno-vectored vaccine [51,52].
In contrast to the BNT162b2 vaccine, a homologous Ad26.COV2.S boost elicited limited, if any boosting of immune responses.Binding antibodies, neutralization, and T-cell responses increased 2-fold or less, and halving the dose resulted in no detectable booster activity.In contrast, boosting with BNT162b2 gave a 4-to 10-fold increase of antibody responses, with the exception of ADCC (discussed below), and 80% of trial participants had increased spike specific CD4+ T-cell responses.Unlike with Ad26.COV2.S, halving the dose of BNT162b2 did not strongly decrease the response to the booster in terms of either elicited binding antibodies, neutralizing antibodies, or CD4+ T-cell responses.Despite the strong boosting effect of BNT162b2, waning of the binding and neutralizing antibody responses was pronounced by 12 weeks post-boost and by week 24, titers had declined close to the relatively high baseline titers.For ADCC, which generally correlates well with binding and neutralization, the absence of boosting by Ad26.COV2.S is expected, given the lack of boosting overall.
However the marginal ADCC boosting observed only in the full-dose BNT162b2 arms (despite increased binding and neutralization) may be a consequence of class switching towards the IgG4 subclasses, which does not effectively mediate ADCC, as recently reported for repeated mRNA vaccination [53,54].
The effects of HIV status on the neutralizing antibody and T-cell responses were moderate, and largely limited to reduced responses observed at baseline in PLWH with HIV viremia (despite the small size of this group).Both binding neutralizing antibody responses to ancestral SARS-CoV-2 were significantly lower in HIV viremic participants at baseline, while there was no difference between aviremic PLWH and HIV-negative participants.CD4+ T-cell responses showed a more marked difference with only 50% of viremic PLWH having detectable spike-specific CD4+ T cells, compared with 100% of aviremic PLWH and 97% of HIV-negative participants.For CD8+ T cells, the fraction of detectable spike-specific cells was 58% for both aviremic PLWH and HIV negative participants.In contrast, only 1 out of 10 HIV viremic participants had a detectable spike-specific CD8+ T-cell response.Interestingly, the median fraction of spike specific CD4+ T cells was significantly higher in aviremic PLWH than the HIV-uninfected participants, perhaps indicative of a different SARS-CoV-2 course of infection in PLWH [27] or interactions of SARS-CoV-2 immunity with HIV-mediated partial immune activation in HIV suppressed PLWH [55].
Post-boost, binding antibodies and neutralization were generally similar between PLWH and HIV negative participants.Even viremic PLWH showed increased binding and neutralizing antibody responses after BNT162b2 vaccination, and these were within the range seen in the other participants.These results are consistent with previous studies that PLWH have good antibody responses to vaccination [19,38,39,56] with the exception of those with CD4+ T cell concentrations than 200 cells/mm 3 [34,36,37,57,58].T-cell responses were more strongly affected by HIV status: they were significantly lower for spike-specific CD4+ T cells in PLWH boosted with a full-dose of BNT162b2.We did not detect an increase in spike-specific CD8+ T-cell responses with full-dose BNT162b2, although the results did not reach statistical significance.The interpretation of the CD4+ T cell results is complicated by the higher median absolute fraction of spike-specific CD4+ T cells in PLWH post-BNT162b2 boost.
Although the Ad26.COV2.S is known to trigger lower responses than BNT162b2, the almost complete absence of boosting by this vaccine in our cohort was striking.We thus assessed whether this was caused by anti-vector immunity.Vaccination with adenovirus 5 vectored vaccines elicits both neutralizing antibody and T-cell immunity to the vector itself, decreasing the efficiency of cellular infection with the vaccine vector and attenuating the vaccine response [44][45][46].Using the samples from this trial and supplementing with other cohorts where a pre-Ad26.COV2.S prime vaccination sample was available, we observed negligible Ad26.COV2.S neutralizing activity in vaccine-naïve donors.However, a single dose of Ad26.COV2.S strongly increases both neutralizing titers against Ad26.COV2.S and the fraction of Ad26-specific CD4+ and CD8+ T cells.Nevertheless, as with other studies of anti-Ad26.COV2.S neutralization, we could not find a clear association between the levels of anti-vector immunity, either neutralizing or T cell, and the degree of immune response post-Ad26.COV2.S boost, as previously reported [47,48].
To conclude, we show that in the context of high levels of hybrid immunity, heterologous boosting with the BNT162b2 mRNA vaccine following a Ad26.COV2.S prime demonstrated superior immunogenicity and was safe and effective in both PLWH and HIV-negative participants, although there was a rapid waning of binding and neutralizing antibody responses.The Ad26.COV2.S homologous boost was also safe but showed highly attenuated immunogenicity relative to BNT162b2, both in antibody and T-cell immunity.While the Ad26.COV2.S vaccine elicited strong anti-vector immunity, we did not find clear evidence that this was the cause of attenuation.
Design and Participants.This study, Booster After Sisonke Study (BaSiS), is an ongoing phase 2, randomized, open-label trial to evaluate the safety and immunogenicity of a booster vaccination in participants who received a single Ad26.COV2.S vaccine through the Sisonke phase 3B implementation study or via the South African National Department of Health COVID-19 vaccine rollout.Participants were healthy adults, who met study eligibility criteria and included participants with well-controlled comorbidities, except for HIV infection where there were no immunological or virological exclusions.Full eligibility criteria are provided in the protocol.We aimed to enrol at least one third of participants as PLWH and at least 10% >55 years of age.Participants were included if they had no SARS-CoV-2 infection at least 28 days prior to randomization.The study was conducted at four research sites in South Africa, two in Johannesburg, one in Durban and one in Cape Town.Randomization.Participants were randomized 1:1:1:1 to one of four booster vaccinations including: Arm A: full-dose Ad26.COV2.S (5x10 10 vp/mL, 0.25 mL); Arm B: half-dose Ad26.COV2.S (2.6x10 10 vp/mL, 0.13 mL); Arm C: full-dose BNT162b2 Comirnaty vaccine (30mcg) and Arm D: half-dose BNT162b2 Comirnaty vaccine (15mcg).No masking was required since the study was open-label.

Figure 2 :
Figure 2: Recorded adverse events in each study arm.Distribution of participants

Figure 3 :
Figure 3: Spike-specific IgG responses over time in the immunogenicity sub-study

Figure 9 :
Figure 9: Humoral and cellular responses in study participants stratified by HIV status.

Table 1 :
Clinical Characteristics of study participantsUnless specified, all data are presented as n (%N); *median and interquartile range (IQR); $ % is of all HIV-infected participants; Viremic individuals were defined as having a HIV-1 viral load (VL) >200 HIV mRNA copies/mm 3 ; 3 participants in the viremic arm C had a missing CD4 count; & Prior SARS-CoV-2 infection was defined by the presence of Nucleocapsid-specific IgG; na: non-applicable. .